Isolation and reconstitution into the bilayer of lipid vesicles or liposomes is one of the most powerful techniques applied to the study of membrane proteins. The simplicity of the resulting model system and the ability to control experimental conditions, allow studies which would be difficult or impossible to perform or interpret using intact cells, viruses, or whole isolated membranes. The structure and function of viral glycoproteins and transport systems, and interactions of membrane receptors with lectins, viruses, and hormones have been investigated using liposomere-constituted membrane proteins. This approach has also proven useful in a variety of immunological studies involving antibody and complement binding, the activities and generation of cytotoxic T lymphocytes, particle uptake by macrophages and the production of subunit vaccines.
Another reason for reconstituting membrane proteins into artificial lipid bilayers is to modify the properties of liposomes. Liposomes have tremendous potential as delivery vehicles in vivo and in vitro owing to their ability to encapsulate, store, and transport materials. However, they have a low efficiency of attachment to cells. Also, a significant proportion of those which do attach are taken up by endocytosis, which results in their contents being delivered to lysosomes. This is desirable in some cases, as in the proposed treatment of some lysosomal storage diseases by liposomally delivered enzymes. However, if the goal is delivery to the cytoplasm or nucleus in a biologically active form, contact with the lysosomes should be avoided.
In enveloped viruses, the delivery of the viral nucleocapsid to the cytoplasm of the cell in a biologically active form is achieved through the interaction of the viral envelope glycoproteins with plasma membrane components. The viral glycoproteins mediate attachment to cell surface receptors and bring about the fusion of the viral envelope with the cell membrane at the surface, or within the low pH environment of the endocytic vesicle. It has been suggested that liposomes containing these biologically active glycoproteins integrated in their lipid bilayer might be superior delivery vesicles. See in this regard Straubinger et al., Cell 32, 1069 (1983) and Volsky and Loyter, FEBS Lett. 92, 190 (1978). It has been shown that reconstituted Sendai virus envelopes can efficiently deliver entrapped molecules to the cytoplasm and nucleus of animal cells. See in this regard Loyter and Volksy in Membrane Reconstitution, Cell Surface Reviews (Poste, G. and Nicholson, G., eds.) Vol. 8, pp. 216-265 (1982).
The preparation of large unilamellar, phospholipid vesicles made by the use of a calcium-EDTA-chelation technique has been described by Papahadjopoulos et al., Biochim. Biophys. Acta 394, 483 (1975).
However, liposomes prepared in accordance with the teaching of this reference have not been heretofor used to reconstitute membrane proteins in biologically active form. Moreover, the efficiency of encapsulation of materials within the liposome is relatively low.